A major impediment to recovery after central nervous system (CNS) injury is the failure of axons to regrow effectively. A variety of extrinsic and intrinsic factors contribute to this problem. Extrinsic factors include inhibitory proteins found i and around the injury site such as those from the glial scar as well as those associated with intact or damaged myelin. Regarding intrinsic factors, a key finding motivating this work is that Dorsal Root Ganglion (DRG) neurons can respond to peripheral injury with changes in gene expression that promote CNS regeneration, even in the inhibitory environment around the injury site. In contrast, CNS neurons typically fail to regenerate axons through such inhibitory regions. This implies that CNS neurons have inherent molecular differences that limit CNS regenerative capacity. The recent discovery of PTEN, SOCS3, KLF4 and KLF7 as important intrinsic regulators of CNS axon regeneration validates this hypothesis. However, the small fraction of CNS axons able to regenerate after injury, even in animals in which these genes have been manipulated, indicates that additional regulators remain to be discovered. The present proposal is to use high throughput technologies to identify genes regulating CNS regeneration by examining two related hypothesis about intrinsic factors. The first is a direct continuation of the hypothesis driving the preceding grant; i.e., that DRG neurons express RNAs that are expressed at significantly lower levels in CNS neurons and allow DRG axon regeneration. The second is that DRG neurons that have experienced a conditioning peripheral lesion express RNAs that allow regeneration and are missing (or very lowly expressed) in lesioned CNS neurons, such as the corticospinal neurons. Aim 1 will identify these molecular differences using RNA-Seq combined with bioinformatics approaches. These methods are effective at identifying rare and potentially novel isoforms, allowing identification of targets that are important but may not be abundant or previously identified; examples include miRNAs and specific transcription factor isoforms. Candidates will be tested using phenotypic analysis in vitro. Aim 2 will use viral vectors to transduce corticospinal tract neurons and test candidates from Aim 1, alone and in combination, using a pyramidotomy model of axonal growth. These experiments will provide novel information about the genes expressed in DRG neurons that allow them regenerate in the injured CNS. The identification of these targets and the testing of multiple candidates both in vitro and in vivo should lead to potential treatments not only for SCI, but also for other CNS disorders such as traumatic brain injury and stroke.